Isolated peptides to treat alpha-synuclein diseases

ABSTRACT

The present invention relates to drug screening methods and methods of preventing neural tissue damage caused by α-synuclein aggregation. These methods are especially useful in the design and development of inhibitors of Lewy body diseases and other synucleinopathies, and further useful in the treatment of such neurodegenerative diseases, particularly Parkinson&#39;s Disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to U.S.application Ser. No. 09/901,187, which was filed on Jul. 9, 2001 nowU.S. Pat. No. 6,780,971. This application also claims benefit of U.S.Provisional Application Nos. 60/217,319 and 60/279,199, which were filedon Jul. 7, 2000 and Mar. 28, 2001 respectively.

FIELD OF THE INVENTION

The present invention relates to drug screening methods and methods ofpreventing neural tissue damage caused by α-synuclein aggregation. Thesemethods are especially useful in the design and development ofinhibitors of Lewy body diseases and other synucleinopathies, andfurther useful in the treatment of such neurodegenerative diseases,particularly Parkinson's Disease.

BACKGROUND OF THE INVENTION

Parkinson's disease (PD) is a common neurodegenerative disorder and wasfirst described by James Parkinson in 1817. The four primary diagnosticsigns of the illness are resting tremor, bradykinesia, muscular rigidityand postural instability. These signs of motor deficiency result fromthe loss of dopaminergic neurons in the nigrostriatal system [Gibb, W.,et al., J. Neurol. Neurosurg. and Psych., 51:745-52 (1988)].

PD is characterized by the formation of Lewy bodies and death ofdopaminergic neurons. [Adams D. et al., Principles of Neurology,874-880, 3rd Edition, McGraw-Hill, NY, (1985)]. The neuropathologicalhallmark of PD is the Lewy body. Lewy bodies are intracytoplasmicinclusions that occur in degenerating neurons which are composed of adense core of filamentous and granular material surrounded by radicallyoriented filaments that have a diameter of 10-20 nm [Goedert, 20 M., etal., Curr. Op. Neurobio., 8:619-32 (1999)]. In general, the causes of PDare not known and there has been vigorous debate over the relative rolesof genetics and environmental factors [Tanner, C., et al., JAMA,281:341-6 (1999)]. Exposure to manganese precipitated a Parkinsoniansyndrome in miners which also includes schizophrenia form behaviors.Some epidemiological studies have found an association betweenindustrial exposure to iron and the incidence of PD [Corell J. M et al.,Toxicol. Appl. Pharmacol., 80:467-72, (1985)], between incidence of PDand blood mercury levels. [Ngim C. H. et al., Neuroepi., 8(3):128-141(1989)] and with death rates from PD and proximity to iron relatedindustrial processed [Rybicki A. et al., Mov Disord., 8(1):87-92(1993)].

α-Synuclein was originally identified as a protein that is upregulatedassociated with neuron outgrowth during the critical period of Zebrafinch song learning [George M., et al., Neuron, 15:361 (1995)].α-Synuclein is a ubiquitous protein that shares significant physical andfunctional homology to the protein chaperone, 14-3-3, and isparticularly abundant in the brain (Ostrerova N. et al, J. Neurosci.,19:5782 (1990); Clayton D. et al., TINS 21:249 (1998)]. α-Synuclein isnormally phosphorylated at serines 87 and 129. (Okochi M. et al, J.Biol. Chem., 275:390 (2000)]. Recent studies showed that mutations inα-synuclein can cause familial PD and that α-synuclein accumulates inLewy bodies. These discoveries suggest that α-synuclein participates inthe pathophysiology of PD. (Spillantini M. et al., Nature, 388:839(1997); Spillantini M. et al, PNAS USA, 95:6469 (1998); Jenner P. et al,Ann. Neurol., 44:S72 (1998)]. The only identified mutations associatedwith familial PD to date are the A53T and A30P mutations in theα-synuclein gene (Goedert, M., et al., Curr. Op. Neurobio., 8:619-32(1999); Papadimitriou, A., et al, Neurology., 52:651-4 (1999);Polymeropoulos, M., et al, Science, 276:1197-9 (1997)]. However, therehas been much circumstantial evidence implicating oxidative stress inthe etiology of the disease (Jenner, P., et al., Annual Neurol.,44:S72-84 (1998)].

A variety of experimental evidence suggests that Lewy bodies interactwith α-synuclein. For example, immunohistochemical studies indicate thatLewy bodies stain strongly for α-synuclein and ubiquitin (Jenner, P., etal., Annual Neurol., 44:S72-84 (1998); Markopoulou, K., et al., Annual.Neurol., 46:374-81 (1999); Spillantini, M., et al., Nature, 388:839-40(1997); and Spillantini, M, et al., Proc. Natl. Acad. Sci. USA,95:6469-73 (1998)]. In vitro experiments using recombinant proteinsuggest that the mutations, A53T and A30P, increase α-synucleinaggregation in comparison with the wild type α-synuclein (Conway, K., etal., Nature Med., 4:1318-20 (1998); Giasson, B., et al., J. Biol. Chem.,274:7619-22 (1999); Hashimoto, M., et. al., Brain Res., 25 799:301-6(1998)].

One of the important questions regarding α-synuclein aggregation andLewy body formation is whether these processes harm the cell. Lewybodies could either be inert tombstone markers that occur in response tofree radical damage, or they might be toxic agents that harm the cell.Examples of both situations exist in the literature. Aggregatedamyloid-β (Aβ) is toxic to neurons, while lipofuscin appears to beinnocuous to cells (Behl, C., et al., Cell 77:817-27 (1994)]. TheHuntington's protein presents an intermediate situation where thetoxicity associated with Huntington's appears to precede aggregation,and aggregation of Huntington's protein might even be protective[Saudou, F., et al., Cell 95:55-66 (1998)]. Our own previous studiesshowed that transient over-expression of α-synuclein is toxic to avariety of cells, including two neuronal cell lines, SK-N-SH and PC12[Ostrerova, N., et al., Neurosci., 19:5782-91 (1999)]. Consistent withthis observation, Masliah and colleagues have recently shown that miceover-expressing α-synuclein show an age-related loss of dopaminergicterminals and motor impairment, which could be indicative of toxicity[Masliah, E. et al., Science 287:1265-1269 (2000)]. These findingssuggest that an increased rate of α-synuclein aggregation mightcontribute to the mechanisms of neurodegeneration in PD and other Lewybody diseases.

Recent studies on transgenic animals also suggest that aggregation ofα-synuclein is harmful to neurons. It was recently reported thatdopaminergic dysfunction occurred in transgenic mice expressing wildtype human α-synuclein [Masliah, E., et at., Science 287:1265-1269(2000)]. Further, it was reported that Drosophila over-expressingα-synuclein exhibited dopaminergic dysfunction and dopaminergic neuronaldeath associated with development of α-synuclein aggregates [Feany, MB,et al., Nature 404:394-8 (2000)]. Evidence suggests that neurons withdopamine develop α-synuclein aggregates and degenerate as theseaggregates development.

Recently, oxidative stress produced by iron and hydrogen peroxide hasbeen shown to induce amyloid-like aggregate formation of α-synuclein invitro [Hashimoto, M., et at., NeuroReport., 10:717-21 (1999); Paik, S.,et al., Biochem. J., 340:821-8 (1999)]. Oxidative stress is thought tocontribute to PD because dopamine, which is a strong free radicalgenerator, is the principle neurotransmitter in the substantia nigra[Chiueh, C., et at., Adv. Neurol., 60:251-8 (1993); Jenner, P. et al.,Ann. Neurol., 25 44:S72-84 (1998)]. In addition, iron, which alsostimulates free radical production, accumulates in the substantia nigrawith age [Jenner, P., et al., Ann. Neurol., 44:S72-84 (1998)]. Iron isdeposited as hemosiderin granules in the cytoplasm, and mitochondriafilled with ferritin granules have been observed in the neuronal andglial cells of the ventorlateral thalamus, caudate and lenticular nucleiand substantia nigra of Parkinsonian brains. [Earle M., J. Neuropathol.Exper. Neurol., 27(1):1-14, (1968); Asenjo A. et al., Rev.Neurologigue., 121 (6):581-92, (1969); Riederer P., et al., J.Neurochem., 52(2):515-20, (1989)]. Thus, the oxidative conditionspresent in the substantia nigra could promote α-synuclein aggregation.However, in the prior art, whether such oxidative conditions actuallypromote α-synuclein aggregation in living neurons is unknown.

A need, therefore, exists for substances that will inhibit Lewy bodyformation, which can be used to treat diseases such as PD. At the sametime, ways to find such substances, through screening assays, are alsoneeded in the art. U.S. Pat. No. 6,184,351, discloses that synucleinaggregation can be induced by continually shaking α-synuclein for longperiods of time at very high concentrations. However, the required highconcentration and continuous shaking is neither physiologic norconducive to the development of screening assays. Moreover, the assaydescribed in the '351 patent does not directly measure α-synucleinaggregation; rather the soluble, unaggregated protein is determined.Further, the '351 assay is directed only to detectingnucleation-affecting agents, and not agents that would inhibitaggregation by other mechanisms. Therefore, the art would benefit frombetter methods for screening drugs useful for the inhibition of Lewybody formation.

SUMMARY OF THE INVENTION

The present invention relates to methods of identifying factors thatlead to the inhibition of the aggregation of α-synuclein in vitro or inliving neurons.

It is an objective of the present invention to provide a method ofidentifying, screening, and modeling pharmaceutical agents capable ofinhibiting the aggregation pathway of α-synuclein. The pharmaceuticalagents are, for instance, cations, small molecules, peptides, peptidemimetic compounds, nucleic acids, complex sugars, such as heparinanalogues, or combinations thereof.

It is also an objective of the present invention to design, make, anduse anti-α-synuclein aggregation drugs for treating a neurodegenerativecondition, such as PD, Alzheimer's disease, diffuse Lewy body disease,mixed AD-PD, multiple system atrophy and Hallervorden-Spatz disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: α-Synuclein increases iron dependent toxicity.

A & B.) BE-M17 cells over-expressing wildtype, A53T or A30P α-synucleinwere treated with varying doses of FeCl₂ for 48 hrs and the viabilitywas determined using the MTT assay (A) or the LDH assay (B).

C.) BE-M17 cells over-expressing wildtype, A53T or A30P α-synuclein weretreated with varying doses of H₂O₂ for 48 hrs and viability wasdetermined using the MTT assay. *p<0.001, +, p<0.01 by ANOVA analysis.

FIG. 2: Iron binds to α-Synuclein:

A.) FeCl₂ quenches the fluorescence emission spectrum by tyrosine inα-synuclein (λex=280 nm). High doses of iron (>10 mM) will quenchtyrosine fluorescence, however proteins that bind iron exhibit quenchingat much lower doses (presumably because the iron is kept near thetyrosine by the protein binding). Tyrosine has a fluorescence emissionspectrum that has a peak emission of 310 nm when excited at 280 nm,respectively.

B.) Dose response curve for iron binding to wildtype and ΔC₁₋₁₃α-synuclein based on fluorescence emissions. A deletion constructlacking the last 27 amino acids of α-synuclein and analyzed binding ofthis construct. A C-terminal deletion construct of α-synuclein ΔC₁₋₁₁₃showed over a 4-fold reduction in iron binding, with an IC₅₀=726 μM(P<0.001) (FIG. 2B).

FIG. 3: Magnesium protects against synuclein aggregation.

A.) Magnesium converts α-synuclein to a conformation that resistsaggregation.

B.) Magnesium inhibits α-synuclein aggregation. a.) Magnesium (0.1 mM)inhibits iron-induced aggregation of recombinant wild type α-synuclein(4 μM, 24 hr incubation). b.) Magnesium also induces α-synucleinaggregation in primary neurons. Higher doses are needed because the cellmembrane poses a barrier to passage of the ions. Similar results areseen in BE-M17 cells over-expressing α-synuclein.

DETAILED DESCRIPTION OF THE INVENTION

Like many other proteins involved in neurodegenerative disease,α-synuclein has a high propensity to oligomerize/aggregate, forming thelarge protein filaments that are deposited in Lewy bodies. In vitro,α-synuclein tends to aggregate over a 3-day period. This rate isincreased 2-3 fold in the mutant forms of α-synuclein, A53T and A30P.Metal ions, oxidative stress and other environmental factors may alsoincrease this rate of aggregation (see below). The present invention isdirected to the development of therapeutic agents that can inhibitα-synuclein oligomerization and thus Lewy body formation andneurodegeneration. Methods by which such agents may be readilyidentified are also contemplated by the present invention.

Thus, one aspect of the present invention relates to methods fordetermining whether an agent is useful in ameliorating the symptoms of aneurological disease that involves the aggregation of α-synuclein aspart of its pathway, which measure the ability of the agent to preventthe aggregation of α-synuclein. One such method determines whether theagent is capable of inhibiting the aggregation pathway of α-synuclein,and comprises adding the agent to a sample containing α-synuclein in thepresence of exogenous iron, and allowing the α-synuclein to aggregate,determining the amount, if any, of aggregation of α-synuclein, and thencomparing that amount with an amount determined in a control sample inwhich the agent is absent. A decrease in the amount of aggregationindicates that the agent is capable of inhibiting the aggregation ofα-synuclein, and thus that the agent would be useful in the treatment ofthe disease. The exogenous iron is added to induce aggregate formation.The range of iron concentration is about 0.1 to 1 mM, preferably 0.1 to1 mM.

The above assay may be performed in vitro with α-synuclein, or in a cellculture system or animal model. For the in vitro assay, to a solution ofα-synuclein (is) are added components that will accelerate aggregation,such as an exogenous source of iron or copper (for example, salts ormetalloproteins), and/or free radical generators, for example dopamineand hydrogen peroxide. A mutant α-synuclein (A30P or A53T) which tendsto have accelerated oligomerization may also be used. Concurrently, theagent to be tested is added, and sufficient time is allowed foraggregation to proceed. Typically, this is about 12 hours, althoughlonger time periods may be observed.

There are several ways of detecting any aggregated α-synuclein, and thepresent invention is not limited hereby. One method exemplified hereinemploys SDS-PAGE and immunoblotting. α-Synuclein oligomers have beenshown to be stable to SDS and, thus, oligomer-banding patterns can beidentified on a blot. A second method of determining the extent ofoligomerization is to stain the oligomers with thioflavine-S andvisualize the filaments by fluorescence microscopy. Thioflavine-S bindsto the β-pleated sheets that are formed upon oligomerization.

One may also use a novel fluorescence anisotropy assay for α-synucleinoligomerization that can be of great utility for higher throughput drugscreening, and which is also a subject of the present invention.Fluorescence anisotropy is a measure of the rotational diffusion of afluorophore in solution. Small molecules rotate more rapidly than largercomplexes, thus as α-synuclein oligomerizes the fluorescence anisotropyof an associated fluorophore would be expected to increase. α-Synucleincan be specifically labeled on the N-terminus with an amine reactivefluorophore at neutral pH, which may be chosen from fluoroscein, pyreneand dansyl derivatives, for example. By monitoring changes in theanisotropy, one can readily determine compounds that can inhibitaggregation because they would be expected to block an increase inanisotropy. See Lakowicz, Joseph R., Principles of FluorescenceSpectroscopy, Chapter 5, pp. 111-153, Plenum Press (NY) (1986), which isincorporated herein by reference.

For a cell culture assay, neuroblastoma cells can be used, such asBE-M17 cells, which are publicly available from the ECACC. In order toinduce α-synuclein aggregation in the cells, the cells are transfectedwith a vector that expresses α-synuclein or one of the mutant variantsthereof (A30P or A53T), thereby upregulating the production ofα-synuclein. This upregulation has the effect of inducing aggregation,which would not otherwise occur. As with the in vitro assay above, Fe²⁺with or without free radical generators (such as hydrogen peroxide anddopamine) is added to the cell culture, and the cells are monitored forintracellular oligomerization over a 12-96 hour period. The aggregationcan be detected by, for instance, histochemical staining withthioflavine-S or labelled anti-α-synuclein antibodies. Alternatively,the cells could be harvested, lysed, separated on an SDS-polyacrylamidegel, and immunoblotted with labeled anti-α-synuclein antibodies. Anotherway to detect the aggregated α-synuclein is by using electronmicroscopy.

Preferably, one or more free radical generators, such as dopamine andhydrogen peroxide, are added together with the iron (Fe²⁺) to the cellculture assays.

Most preferably, only Fe²⁺ and hydrogen peroxide are added as theinducers of aggregation (but for cells with the A53T mutant, no freeradicals are necessary).

By the term “labelled” is meant labelling with any substance typicallyused in biochemical assays of this sort, and includes, for example,radioactive, enzymatic, chemiluminescent, and fluorescent labels.Preferably, the label is enzymatic, and most preferably the label is aperoxidase.

In a cell culture assay using human BE-M17 neuroblastoma cells thatover-express wildtype, A53T mutant or A30P mutant α-synuclein, theamount of aggregation occurring in the cells is dependent on the amountof α-synuclein expressed and the type of α-synuclein expressed, with theamount of α-synuclein aggregation following a rank order ofA53T>A30P>wildtype>untransfected. In addition to stimulating aggregateformation, α-synuclein also appears to induce toxicity. BE-M17neuroblastoma cells over-expressing α-synuclein show up to a 4-foldincrease in vulnerability to toxicity induced by iron. The vulnerabilityfollows the same rank order as for aggregation. These data suggest thatα-synuclein acts in concert with iron and dopamine to induce theformation of Lewy body pathology in PD and cell death in PD.

In another aspect of the present invention is a method of inhibiting theformation of α-synuclein aggregates. More specifically, this method isfor the 20′ treatment of Lewy body diseases, such as Parkinson'sdisease, whereby Lewy body formation is controlled (or lessened) byinhibiting α-synuclein aggregation. That is, the present inventioncontemplates a method for treating a neurodegenerative disease thatinvolves the formation of Lewy bodies, which comprises administering toa patient in need thereof one or more agents that inhibit the formationof α-synuclein aggregates, whereby the presence of Lewy bodies remainsthe same or, more preferably, is reduced. The particulars of suchtreatment depend on the therapeutic agent involved, the particulardisease and its stage of progress, and other factors such asbioavailability, etc., which are all factors that the skilledpractitioner can take into account when determining dosages, routes ofadministration, and length of treatment, and the present invention isnot limited hereby.

In another aspect of the present invention are therapeutic agents forthe treatment of Lewy body diseases, which have been discovered by thepresent inventors. In particular, the present inventors found that Mg²⁺inhibits the aggregation of α-synuclein. More specifically, theinventors found that both iron and magnesium bind to α-synuclein, butexert opposing actions on α-synuclein aggregation. Iron increasesα-synuclein aggregation, whereas magnesium inhibits α-synucleinaggregation. In addition to decreasing aggregation of wildtypeα-synuclein in cell culture, magnesium also decreases formation ofvisible neuronal inclusions and protects against neurotoxicity. Thesedata are indicative of a therapeutic role for magnesium in the treatmentof a Lewy body disease, such as PD, in which the progression of thedisease can be at least slowed and likely reversed.

It is known that iron levels are increased in brains of patients withPD, whereas magnesium levels are reduced, but the role that these metalsplay in the pathophysiology of PD has never been elucidated. See, forinstance, Durlach et al., Magnes. Res. 11:2542 (1998). The prior artdoes not suggest that regulating the balance of iron and magnesium inthe brain would have an affect on Lewy body formation or diseaseprogression. U.S. Pat. Nos. 4,985,256; 4,837,164; and 5,004,615 to Glickdisclose administering magnesium to improve memory in dementias, amongwhich is mentioned ALS-PD (“amyotropic lateral sclerosis andParkinsonism complex”) This is a disease recognized in Guam, andinvolves high levels of aluminum and low levels of magnesium in braincells of those affected. However, the condition is very different fromParkinson's disease, particularly with respect to Lewy body formation,which is absent in ALS-PD. Moreover, The purpose for which Glicksuggests supplementing magnesium is to raise magnesium to normal levelsin the recited demtentias, not to interfere with α-synuclein aggregaton.It was not until the present invention that it was shown that magnesiumhas an affect on α-synuclein aggregation.

Therefore, as a preferred embodiment, Mg²⁺ is administered to a mammalin need of treatment of a Lewy body disease. The Mg²⁺ may be in anynon-toxic, pharmaceutically acceptable form. Preferably, the magnesiumis administered as magnesium sulphate, magnesium phosphate, magnesiumgluconate, magnesium oxide, magnesium hydroxide, magnesium chloride,magnesium carbonate, or combinations thereof. For parenteraladministration, most preferable is magnesium sulphate.

As used herein, the term “administration” refers to the application ordelivery of a drug to a mammal. This term is intended to include anymeans of administration which accomplishes the application or deliveryof drug, but preferred are those means of administration that target themagnesium to the brain tissue involved. The term is also intended toinclude any means necessary to accomplish such administration, such as asugar loading procedure to enable a drug to cross the blood-brainbarrier, if required.

The dosage of magnesium is determinable by the skilled practitioner, forinstance by referring to treatment regimens of other conditions withmagnesium salts. Magnesium should not be administered such that toxiclevels are attained. The present inventors have found experimentallythat the magnesium required to inhibit α-synuclein aggregation in vitrois within the range commonly employed in clinical protocols usingmagnesium (for example, eclampsia of pregnancy), i.e., 0.5-2 mM MgSO₄.Plasma levels in a normal adult human are between 1.5 and 2 mEq perliter, about one third of which is bound to protein. In hypermagnesemia,which is primarily due to renal insufficiency, excess magnesium resultsin results in depression of the central nervous system and theperipheral neuromuscular junction. When plasma levels begin to exceed 4mEq per liter, deep tendon reflexes become decreased or absent, at whichpoint respiratory paralysis is a risk. Therefore, in the treatment withmagnesium salts in accordance with the present invention, appropriatemonitoring of magnesium levels should be performed.

Magnesium salts have been used therapeutically in humans for years. Forinstance, many antacids are comprised of magnesium salts, specificallycitrate and sulfate salts. Thus, such salts are contemplated as usefulif administration of the magnesium is gastrointestinally. For parenteraladministration (intramuscular or intravenous), which is preferred in thepresent invention because higher systemic levels can be attained,magnesium sulfate is the salt of choice. Such administration has foryears been employed in the treatment of seizures associated witheclampsia of pregnancy, in which an initial and sustaining dosage, baseon body weight, are given. See Flowers et al., Obstet. Gyn., 19:315-327(1962), the contents of which are incorporated herein by reference, fora dosage schedule for toxemia of pregnancy. Such a regimen is applicableto the treatment of a Lewy body disease according to the presentinvention as well, with plasma concentrations also within the 3-6 mg.%range. However, in the case of a progressive neurodegenerative Lewy bodydisease, such as PD, the regimen may span the remainder of life.

The present inventors also discovered that other agents useful fortreating Lewy body diseases and synucleinopathies (diseases that haveα-synuclein involved in their pathways, but not necessarily Lewy bodies)are peptides, or derivatives thereof, that bind to α-synuclein andinhibit the aggregation thereof. These peptides, alone or incomposition, can be used to treat a subject with a neurodegenerativedisease that displays Lewy body pathology. While not being bound by anyparticular theory, it is believed that the binding of the peptide(s) toα-synuclein somehow prevents the α-synuclein from aggregating, perhapsby interfering with the role of Fe²⁺ in the aggregation.

Such peptides can be found, for instance, by using phage display toselect for those phage expressing a peptide on their surface that bindsto α-synuclein, and determining what the peptide is. The inhibitoryeffect of that peptide, or any peptide for that matter, can also betested to determine its usefulness as an agent for treating a Lewy bodydisease or synucleinopathy by using assays such as those disclosedherein or as disclosed in U.S. Pat. No. 6,184,351, which is incorporatedherein by reference. Such peptides have been selected for their abilityto bind to the C-terminal (approx. amino acids 113-140), and the NACportion (approx. amino acids 61-87) of α-synuclein, because this regionis thought to be involved with the aggregation process. Several peptidesthat bind to the C-terminal portion are as follows (all peptides aregiven in 5′ to 3′ order) (1) WRQTRKD (SEQ ID NO: 1); (2) HYAKNPI (SEQ IDNO: 2); (3) ATINKSL (SEQ ID NO: 3); (4) RRRGMAI (SEQ ID NO: 4); (5)THRLPSR (SEQ ID NO: 5); and (6) TKHGPRK (SEQ ID NO: 6). Several peptidesthat bind to the NAC portion are: (1) SLKRLPK (SEQ ID NO: 7); (2)RLRGRNQ (SEQ ID NO: 8); (3) WPFHHHR (SEQ ID NO: 9); (4) HLYHHKT (SEQ IDNO: 10); (5) THIHHPS (SEQ ID NO: 11); and (6) MMMNMRL (SEQ ID NO: 12).The NAC portion was chosen because this piece of the protein has beenfound to aggregate in amyloid plaques in Alzheiiner's disease.Particularly preferred, because of stronger binding properties, areTHRLPSR (SEQ ID NO: 5); SLKRLPK (SEQ ID NO: 7); THIHHPS (SEQ ID NO: 11)and MMMMMRL (SEQ ID NO: 12). Most preferred is the peptide SLKRLPK (SEQID NO: 7).

The peptides can be made by methods well known in the peptide synthesisart, and variants or analogs can also be made according to establishedprinciples. For example, one may wish to increase the therapeuticefficacy of the peptide, change the hydrophobicity/hydrophilicity, orreduce unwanted side effects. The peptide structure can be changed bythe addition of functional groups, such as for targeting the compound tothe neural tissues affected by the particular Lewy body disease. Also,substitutions of one or more amino acids of the peptide may be made, andthese will typically involve conservative substitutions, i.e.,substitutions that retain a property of the original amino acid, such ascharge, etc. Examples of such conservative substitutions are ones madebetween (1) M, I, L and V; (2) F, Y, and W; (3) K, R, and H; (4) A andG; (5) S and T; (6) Q and N; and (7) E and D. The changes to the peptideneed not be conservative; the altered peptide can always be tested forefficacy in aggregation inhibition by the assays mentioned previously.

Preferably, the peptides are resistant to proteolytic digestion. Suchpeptides can be made protease resistant by the presence of D-amino acidsor by the replacement of susceptible peptide bonds with non-hydrolyzablebonds. Many such bonds, and how to introduce them, are known in the art.They include -psi[CH₂NH]— (reduced amide peptide bonds); -psi[COCH₂]—(ketomethylene peptide bonds); -psi[CH(CN)NH]— ((cyanomethylene)aminopeptide bonds); -psi[CH₂CH(OH)]— (hydroxyethylene peptide bonds);-psi[CH₂O]— (peptide bonds); and -psi[CH₂S]— (thiomethylene peptidebonds). The peptides can also be constructed with end modifications,such as an amide at the C-terminus or an acetyl group at the N-terminus,which will make them resistant to proteolytic digestion.

Non-peptide (peptide mimetic) analogs are compounds in which one or moreof the residues in the peptide is (are) replaced by a non-peptidemoiety. The non-peptide moieties should allow the peptide to retain itsnatural, or more bioactive, conformation. See Nachman et al., Regul.Pept. 57:359-370 (1995) for methods to prepare nonpeptide mimeticcompounds.

The peptides may be modified by conjugating them to a compound whichfacilitates their transport across the blood brain barrier. Suchcompounds, for example, may be selected from fatty acids, cationizedantibodies or albumin, and the amphiphilic drug-oligomer conjugatesdisclosed in WO 00/09073, which is incorporated herein by reference.Alternatively, the peptides can be administered with a compound orcompounds which facilitate transport across the blood brain barrier, butwhich is not conjugated to the peptide. For examples of such compounds,see U.S. Pat. Nos. 5,112,596 and 5,268,164, which are both incorporatedherein by reference.

By the term “peptide” is meant any of the foregoing peptides andderivatives thereof, and is not limited in the number of amino acids.Thus, a “peptide” could include a polypeptide.

Compositions, particularly pharmaceutical compositions, comprising oneor more of the peptides also form part of the present invention. Atherapeutically effective amount of the peptide(s) (one which willameliorate disease progression) may be combined with anypharmaceutically and/or physiologically acceptable carrier, such asaqueous solutions, salts, buffers, stabilizers, solubilizers, fillers,diluents, and other known substances, depending on the route ofadministration. The compositions may be prepared in any of a variety offorms suitable for the desired mode of administration. For example,pharmaceutical compositions may be prepared in the form of tablets,powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as solids or in liquid media), soft-gel and hard-gel capsules,suppositories, sterile injectable solutions, sterile packaged powders,and the like. Similarly, the carrier or diluent may include time-delayor time-release material known in the art, such as glyceryl monostearateor glyceryl distearate alone or with a wax, ethylcellulose,hydroxypropylmethylcellulose, methylmethacrylate and the like. Properformulation is dependent upon the route of administration chosen. Forinjection, the agents of the invention maybe formulated into aqueoussolutions, preferably in physiologically compatible buffers such asHank's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art. For oral administration, the compounds can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers known in the art. The peptides maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit-dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. See Remington's PharmaceuticalSciences, 18^(th) Edition, Mack Publishing Co. (1990), which isincorporated in its entirety herein by reference, for a comprehensivelist of formulations.

One or more of the peptides can be administered to a mammal in need oftreatment of a Lewy body disease, in a manner conventionally employedand determined on an individual basis by the practitioner. Preferably,the peptides are administered by injection or gradual infusion overtime. Other routes of administration that may be used are oral,intravenous, intracranial, intraperitoneal, intramuscular, intracavity,intrarespiratory, subcutaneous, transdermal, or liposome-mediateddelivery. Delivery to specifically affected neuronal cells can also beaccomplished using targeted delivery methods, which include conjugationto a molecule that selectively binds to the cells or envelopment inliposomes that contain a targeting molecule which will bind to theaffected cells. Such methods for targeting are well known in the art,for instance in U.S. Pat. No. 5,391,723, which is incorporated herein byreference. In addition, some liposomes are commercially available.

In a preferred embodiment, concomitant administration of the peptide(s)with magnesium therapy as set forth above is envisioned. The combinedtherapy is likely to accelerate the recovery, and reach a more completelevel of recovery. Once the recovery has reached the stage of an absenceof further loss of clinical function, co-administration of magnesium mayallow for a lower dose of peptide to be used for maintenance.

The subjects of therapeutic treatment may be humans, or animals such asrodents, dogs, cats, drosophila and C. elegans. Such animals may beuseful human model systems.

Finally, kit for use in performing an assay to test the affects ofvarious substances on the aggregation of α-synuclein, is alsocontemplated. The test would comprise at a minimum lyophilizedα-synuclein, iron or copper salt, and a buffer, such as PBS or Trisbuffer (at physiological pH). The kit could also comprise a free radicalgenerator, such as dopamine or hydrogen peroxide. Preferably, the kitcomprises iron chloride. For the free radical generator, hydrogenperoxide is preferred.

This invention is illustrated in the Examples that follow. Theseexamples are set forth to aid in understanding of the invention but arenot intended to, and should not be construed to, limit in any way theinvention as set forth in the appended claims.

The following materials and methods were used in the examples herein.

Cloning, Overexpression, and Purification of α-Synuclein:

α-Synuclein (wildtype, A53T and A30P) was cloned into the NotI site ofpcDNA3. The sequence of each construct was confirmed by DNA sequencing.For production of recombinant protein, α-synuclein was inserted into theNcoI/NotI site of the ProEx His 6 (SEQ ID NO: 13) vector (GIBCO/BRL). Togenerate recombinant α-synuclein, Bper (Pierce) reagent was used tosolubilize the recombinant αsynuclein from the IPTG-induced bacteriallysates, which were then passed over a nickel-agarose affinity column,washed and eluted with imidazole according to the manufacturer'sdirections (GIBCO/BRL). Following purification, the His-6 tag (SEQ IDNO: 13) was cleaved with TEV protease and removed by passing through anickel-agarose column. Antibodies used include: polyclonal antiα-synuclein (SC1, 1:2000 for immunoblotting and 1:500 forimmunocytochemistry against human α-synuclein, residues 116-131,sequence =MPVDPDNEAYEMPSEE) (SEQ ID NO: 14), monoclonal antiα-synuclein-1 (1:1000, Transduction Labs), polyclonal rabbitanti-ubiquitin (1:1000 for immunoblotting and 1:500 forimmunocytochemistry, Dako).

Phage display: Incubating a library of phage that is all identicalexcept for a sequence of 7 amino acids that are presented on the coatand randomly differ from phage to phage. Phage which express a sequencethat specifically binds to α-synuclein are then isolated, cloned, andthe DNA sequenced to determined the amino acid sequence. The 7 aminoacid peptide is then synthesized and tested directly.

Cell Culture: Cells were grown in OPTIMEM (Gibco/BRL) supplemented with10% FBS, non-essential amino acids, sodium pyruvate and 500 μg/ml G418,as needed. G418 was used for selection.

Immunoblotting: Cells were harvested with SDS lysis solution (2% SDS, 10mM Tris, pH 7.4, 2 mM β-glycerol phosphate, 1 μM AEBSF). The amount ofprotein was determined using the BCA assay (Pierce), 5-30 mg per lanewas run on 14% SDS polyacrylamide gels and transferred to nitrocellulose(200 mAmp, 12 hrs). The nitrocellulose was then incubated 1 hr in 5%milk/PBS, washed, incubated overnight in 10 antibody, washed, thenincubated 3 hrs in peroxidase coupled 2° antibody and developed withchemiluminescent reagent (NEN).

Cell fractionation: For cell fractionation the cells were harvested inbuffer containing 20 mM Tris, pH 7.4, 2 mM EDTA, 0.25 M sucrose, and 20μg/mL protease inhibitor cocktail (Sigma). The cell lysate was sonicatedand centrifuged at 100,000×g at 4° C. for 1 hr.

MTT and LDH toxicity assay: Cells were plated in 96 well dishes at 5000cells/well in 100 μl growth medium. For the MTT assay, viabilityfollowing 48 hrs of pharmacological treatment was analyzed by adding 0.5mg/ml MTT (MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) and incubating at 37° C. for 3 hrs. Lysis buffer (100 μl of 20%SDS in 50% N,N-dimethylformamide) was then added and the plates are readat 540 nm after 24 hrs. For the lactate dehydrogenase (LDH) assay,viability following 24 hrs of pharmacological treatment was analyzedusing MTS reagent and the Cytox 96 kit (Promega) according tomanufacturer's directions.

Thioflavine-S Histochemistry: Cells were fixed 30 min in 4%paraformaldehyde. Following two PBS washes, the cells were incubatedwith 0.5% Thioflavine-S for 8 min, washed three times in 80% ethanol,washed once in H₂O and then mounted.

Electron Microscopy: Cells were detached by scraping, spun down andfixed in 2% glutaraldehyde for 2 hrs at 4° C. and then post-fixed in 1%osmium tetroxide for 1 hr at 4° C. The samples were dehydrated, embeddedin epoxy resin (Electron Microscopy Science, Fort Washington, Pa.), andcut into 70 nm sections for microscopy. The sections were thenpost-stained with 5% uranyl acetate, and Reynolds lead citrate. Sampleswere viewed with a Hitachi H-600 transmission electron microscope at 75kV.

Iron Staining: Cells were fixed 30 min in 4% paraformaldehyde, followedby two PBS washes, stained using the Accustain® Iron Stain according tomanufacturer's directions (Sigma).

Immunohistochemistry: For light microscopy, cells are fixed with 4%paraformaldehyde, washed, permeabilized by incubation for 30 min with0.2% Triton-X100, blocked with 5% dry milk/1% goat serum/PBS, washed andthen incubated overnight in 1° antibody (1:500). Development is with anABC kit and 3′,3′-diaminobenzidine as per manufacturer's directions(Vector, Burlingame, Calif.).

EXAMPLES Example 1 Iron and Free Radicals Stimulate α-SynucleinAggregation

To test whether A53T and A30P mutations in α-synuclein increase thetendency of α-synuclein to aggregate in neurons, α-synuclein aggregationwas determined in human BE-M17 neuroblastoma cells that were stablytransfected with wild type, A53T or A30P α-synuclein. Each cell line wastreated for 48 hours with freshly prepared FeCl₂ (1 or 10 mM), thenharvested, homogenized and fractionated into membrane and cytoplasmiccomponents. The membrane (5 μg/lane) and cytoplasmic (20 μg/lane)components were immunoblotted with monoclonal anti-α-synuclein antibody.

Aggregates of α-synuclein were evident in the membrane fraction, but notin the cytoplasmic fraction. Treatment of the A53T-expressing cell linewith FeCl₂ induced dose-dependent formation of heterogeneous highmolecular weight-α-synuclein aggregates that migrated in the stackinggel. A large amount of anti-α-synuclein immunoreactivity was alsoapparent in the upper portions of the separating gel, in the range of45-200 kDa. Because these bands are significantly larger than monomericα-synuclein, which has a mass of 19 kDa, these bands might alsorepresent α-synuclein polymers and aggregates. For instance, the bandsat 38 and 57 kDa have sizes consistent with dimers and trimers ofα-synuclein. Immunoblots done with a different antibody, a polyclonalanti-α-synuclein antibody, also showed aggregate production under thesame conditions. Treatment of the other cell lines (untransfected,wildtype and A30P) did not induce α-synuclein aggregates within the 48hr time frame examined.

To determine the effect of increasing the duration of exposure to ironon aggregation of α-synuclein at lower doses of iron, BE-M17 cells whichare over-expressing A53T or wildtype α-synuclein were exposed to FeCl₂for 4 days. Under these conditions, doses as low as 0.3 μM FeCl₂produced detectable aggregation of α-synuclein in cells expressing A53Tα-synuclein. Interestingly, these longer conditions also inducedaggregation of wild type α-synuclein. In contrast, no aggregation ofactin was observed, which suggests that aggregation is selective forα-synuclein.

To determine how the pattern of α-synuclein aggregation in the BE-M17cells compares to that occurring in human brain, the response ofα-synuclein in human cortical brain homogenates (from a neurologicallynormal donor) to iron exposure was performed, in vitro. The pattern ofα-synuclein aggregation in the membrane fraction of the cortical brainhomogenate following treatment for 24 hrs with 500 μM dopamine, 10 mMFeCl₂ and protease inhibitors (dopamine was added as an oxidant, asdescribed below) was similar to that seen in the BE-M17 cells. Thissuggests that α-synuclein in BE-M17 cells and in human brain exhibitsimilar aggregation patterns in response to iron, and both share astrong tendency to aggregate. Further, it was observed that highermolecular weight α-synuclein immunoreactivity occasionally appeared inseparating gels of immunoblots of α-synuclein cell lysates from cellsgrown under basal conditions. However, aggregates that migrated in thestacking gel only occurred after treatment with iron and were neverobserved in any of the cell lines under basal conditions. This supportsprior work done with recombinant α-synuclein in vitro indicating thatthe mutant forms of α-synuclein have a strong tendency to oligomerize[Conway, K A, et al., PNAS USA, 97:571-576 (2000)]. However, migrationof aggregates in the stacking gel might be a stricter test of aggregateformation than migration in the separating gel.

Studies showed that iron might promote protein aggregation by increasingfree radical formation through the Fenton reaction [Wolozin, B., et at.,Arch. Neurol. 57:793-796 (2000)]. If so, adding free radical generators,such as hydrogen peroxide or dopamine, along with the iron mightincrease the amount of α-synuclein aggregation. To further determinethat oxidation enhanced iron-induced aggregation of α-synuclein, BE-M17cells over-expressing A30P or wildtype α-synuclein were treated for 48hrs with 10 mM FeCl₂ plus varying concentrations of dopamine (0.5, 50,and 500 μM). Cells were used to express A30P or wild type α-synucleinover 48: hrs because they do not form aggregates under these conditions,unlike cells over-expressing A53T α-synuclein. As expected, BE-M17 cellsover-expressing A30P or wild type α-synuclein treated with 10 mM FeCl₂alone did not induce any aggregation. Similarly, treatment with 5, 50 or500 μM dopamine alone did not induce formation of large α-synucleinaggregates that migrate in the stacking gel. However, combining 10 mMFeCl₂ with 50 or 500 μM dopamine induced formation of large α-synucleinaggregates. These data show that the combination of oxidants and ironcan exert additive effects on α-synuclein aggregation.

Two separate experiments indicated that the aggregation observed was ageneral property of α-synuclein, rather than an artifact resulting fromuse of clonal cell lines. BE-M17 cells that were transiently transfectedwith A53T α-synuclein cDNA and then treated with 10 mM FeCl₂ plus 100 μMH₂O₂ for 72 hrs developed aggregates similar to those seen in the BE-M17cell lines stably over-expressing α-synuclein In addition, primary ratcortical neurons also showed a strong tendency to develop aggregates,requiring only 60 hrs of treatment with 0.1 mM FeCl₂ and 50 μM dopamineto induce formation of aggregates. Thus, the aggregation was a result ofa biophysical property of α-synuclein, rather than being an artifactspecific to particular clonal cell lines.

Example 2 A53T and A30P α-Synuclein Aggregates Contain Ubiguitin

In PD, Lewy bodies have also been shown to contain large amounts ofubiquitin [Dickson, D., et al., Brain Path., 9:721-32 (1999); Gibb, W.,et al., J. Neurol. Neurosurg. and Psych. 51:745-52 (1988)]. To determinewhether aggregation of ubiquitin also occurred along with α-synucleinaggregation, lysates (membrane fractions) were taken from the BE-M17cells expressing A30P α-synuclein that had been treated with FeCl₂ anddopamine (see Example 1, 48 hr treatment), and were immunoblotted withanti-ubiquitin antibody. The ubiquitin aggregates that accumulated underthese conditions stained strongly for ubiquitin. Aggregates thataccumulated in cells expressing A53T α-synuclein or wild typeα-synuclein after being treated with either FeCl₂ alone, FeCl₂ plushydrogen peroxide or FeCl₂ plus dopamine also contained ubiquitin. Theamount of aggregated ubiquitin generally paralleled the amount ofα-synuclein. The presence of ubiquitin in aggregates did not appear toresult from increased ubiquitin expression because immunoblots of totallysates showed that the total amount of ubiquitin and actin was notsignificantly different between cell lines over-expressing α-synucleinand control cells. These data show that aggregates of α-synuclein thatform in neurons in response to iron treatment are ubiquinated.

Example 3 α-Synuclein Aggregates Form Visible Inclusions Evident byThioflavine-S Histochemistry and Electron Microscopy

Thioflavine-S histochemistry and electron microscopy were used toexamine the aggregates that formed in response to treatment with ironand hydrogen peroxide. Cells from each line (BE-M17: untransfected, wildtype, A30P and A53T a.-synuclein) were treated with 10 mM FeCl₂ or 10 mMFeCl₂ plus 100 μM H₂O₂ for 72 hrs to induce formation of α-synucleinpositive inclusions. The aggregates that formed were observed to bindthioflavine-S, which indicates the presence of β-pleated sheetstructures. The size and number of thioflavine-S-positive aggregatesparalleled the results seen by immunoblotting. The wildtype, A30P andA53T α-synuclein cell lines each showed significant accumulations ofprotein aggregates after treatment for 72 hrs with. 10 mM FeCl₂ plus 100μM H₂O₂. In contrast, the untransfected line showed no accumulation ofthioflavine-S positive aggregates. When only 10 mM FeCl₂ was used totreat the cells, thioflavine-S positive inclusions were observed only inA53T α-synuclein expressing cells. Electron microscopy was used toexamine the aggregates. Cells that express A53T α-synuclein or expressempty vector were treated with 10 mM FeCl₂ plus 100 μM H₂O₂ for 72 hrsand then prepared for electron microscopy. Cytoplasmic inclusions wereapparent in the A53T-expressing cells, but not in the vector-transfectedcells. The inclusions contained mixtures of fibrillar and amorphousmaterial. The fibrils had an approximate diameter of 10 nm and a lengthof up to 10 μm. Although treatment with 10 mM FeCl₂ and 100 μM hydrogenperoxide was toxic to many cells, some cells containing aggregates hadboth fibrillar deposits and organelles that were intact, which suggeststhat aggregation can occur in living cells. The presence of bothfibrillar and amorphous material in the aggregates has been observed inaggregates present in transgenic animals over-expressing α-synuclein[Feany, MB, et al., Nature 404:394-8 (2000); Masliah, E., et al.,Science 287:1265-1269 (2000)].

Example 4 α-Synuclein Aggregates Form Visible Inclusions Evident byImmunohistochemistry

Inclusion formation was tested by immunocytochemistry. Cells thatexpress A53T α-synuclein were treated with 10 mM FeCl₂ plus 100 μM H₂O₂for 72 hrs, fixed and then examined with antibodies to ubiquitin andα-synuclein using peroxidase immunohistochemistry. Peroxidase stainingwas used because fluorescent chromagens, such as FITC or rhodamine,exhibited strong non-specific binding to the cells due to the treatmentwith iron. Immunohistochemistry with both the anti-α-synuclein andanti-ubiquitin antibodies showed uniform staining throughout thecytoplasm of the cells under basal conditions. Following treatment with10 mM FeCl₂ plus 100 μM H₂O₂ for 72 hrs, the staining became lessuniform. Cells stained with anti-α-synuclein antibody often displayedseveral large darkly stained reactive foci and multiple, small punctuatefoci in each cell. Staining with ubiquitin was also present but showedfewer foci per cell. Immunocytochemistry performed with the pre-immunerabbit serum instead of primary antibody showed no reactivity undereither basal or treated conditions.

Example 5 α-Synuclein Aggregation Occurs in Viable Cells

To determine the toxicity effect of FeCl₂ to the cell, and therelationship between α-synuclein aggregation and cell death, aggregateformation and cell viability in the presence of high and lowconcentrations of FeCl₂ was performed. As described above (Examples 3 &4), treatment with 10 mM FeCl₂ plus 100 μM H₂O₂ for 72 hrs induced, inmost non-transfected cells, formation of α-synuclein aggregates.Accordingly, these conditions also killed most of the cells (>90% celldeath).

In contrast, A53T-expressing BE-M17 cells treated with 0.3 mM FeCl₂ plus100 μM H₂O₂ for 96 hrs exhibited much less toxicity, yet still formedα-synuclein aggregates. Parallel sets of cells were processed forimmunocytochemistry with antibodies to α-synuclein (SC1) and ubiquitin,or processed to measure viability using the trypan blue exclusion assay.Untreated cells showed little toxicity with 3.7±1.0% being permeable totrypan blue. The small amount of cell death present might have beencaused by the trypsinization/trituration step used to dislodge thecells. Treatment with 0.3 mM FeCl₂ plus 100 μM H₂O₂ killed some cells(12.1±2.3% trypan blue positive), but the large majority of cells(87.9%) remained viable. Immunocytochemistry with anti-α-synucleinantibody showed that 21.0±3.5% of the cells had visible aggregates.Immunocytochemistry with anti-ubiquitin antibody suggested that theaggregates could contain ubiquitin, and that the aggregates containedmaterial that had a β-pleated sheet structure that stained withthioflavine-S. Interestingly, diffuse thioflavine-S reactivity was alsoevident in these cells, suggesting that dispersed ‘micro-aggregates’ ofα-synuclein might also form under the mild conditions.

Thus, the α-synuclein aggregates that formed under mild conditionsreacted with the same antibodies and stains as aggregates that formedunder harsher conditions. Moreover, the observation that the percentageof cells displaying α-synuclein aggregates was greater than thepercentage of cells showing evidence of death suggests that many of thecells developing α-synuclein aggregates are viable.

The observation that aggregation of α-synuclein can occur in viablecells also suggests that aggregation of α-synuclein precedes cell death.

Example 6 Over-Expression of α-Synuclein Increases Free-Radical MediatedToxicity

Despite the fact that α-synuclein aggregates can form in viable cells,it is possible that aggregation of α-synuclein might represent aninitial step in the induction of toxicity. The previous results showedthat α-synuclein was toxic to some cells when transiently over-expressed(Ostrerova, N., et al., J. Neurosci., 19:5782-91 (1999)]. However,α-synuclein is not acutely toxic to all cells, such as BE-M17 cells.α-Synuclein is tolerated well enough to allow over-expression intransgenic animals (Feany, MB, et al., Nature 404:394-8 (2000); Masliah,E., et al., Science 287: 1265-1269 (2000)]. Although α-synuclein is notacutely toxic to BE-M17 cells under basal conditions, it was possiblethat α-synuclein might be toxic under other conditions, such asconditions linked to formation of aggregates.

To determine if the conditions that produce α-synuclein aggregation alsoproduce toxicity, the vulnerability of each cell line to iron and/orhydrogen peroxide-mediated toxicity was tested. Each cell line(untransfected, wild type, A53T and A30P α-synuclein) was treated withvarying doses of FeCl₂, H₂O₂ or FeCl₂+H₂O₂ and the amount of toxicitywas determined by MTT assay. BE-M17 cells over-expressing all forms ofα-synuclein showed increased vulnerability to iron-mediated toxicity(FIG. 1A). Over-expression of A53T α-synuclein had the greatest effecton toxicity, reducing the LD₅₀ of FeCl₂ over 75% (FIG. 1A).Over-expression of A30P or wild type α-synuclein constructs reduced theLD₅₀ values by ˜50%, although the amount of toxicity seen inA30P-expressing cells in response to low levels of iron was generallygreater than toxicity seen with wildtype α-synuclein (FIG. 1A). Toconfirm that over-expression of α-synuclein increased the vulnerabilityof the neuroblastoma cells to iron, iron-induced toxicity was testedusing a LDH assay (FIG. 1B). The LDH assay confirmed thatover-expression of the α-synuclein constructs increases iron-inducedtoxicity (FIG. 1B).

MTT assays of cell lines over-expressing α-synuclein (wildtype, A53T orA30P) also revealed increased toxicity after treatment with FeCl₂+H₂O₂(FIG. 1C, dark gray bars). Interestingly, over-expressing α-synuclein(wildtype, A53T or A30P) did not increase the vulnerability to H₂O₂alone, and conferred modest protection to the neuroblastoma cells (FIG.1C). These data demonstrated that increased levels of either wildtype ormutant α-synuclein can be toxic to neurons grown in cell culture underselective conditions. The data suggest that α-synuclein renders thecells particularly vulnerability to iron-mediated toxicity. Theselective vulnerability to iron could result from a tendency ofα-synuclein to sequester iron in α-synuclein aggregates.

Untransfected or A53T-α-synuclein expressing BE-M17 cells were treatedwith 10 mM FeCl₂ and 100 uM H₂O₂ for 48 hrs, then were fixed and stainedfor iron. The cells expressing A53T α-synuclein had much higher ironcontent than the untransfected cells after treatment. Sequestration ofiron due to α-synuclein would increase free radical production via theFenton reaction, particularly in cells exposed to free radicalgenerators, such as hydrogen peroxide or dopamine. However, in theabsence of iron, no Fenton reaction occurs and the α-synuclein isinnocuous.

Example 7 Iron Binds to α-Synuclein

Because iron binds to other proteins that form aggregates and iron isknown to accumulate in Parkinson's disease, iron might bind directly toα-synuclein. To confirm this hypothesis, the interaction of iron withα-synuclein was tested using tyrosine fluorescence as an indicator ofiron binding. High doses of iron (>10 mM) will quench tyrosinefluorescence, however proteins that bind iron exhibit quenching at muchlower doses (presumably because the iron is kept near the tyrosine bythe protein binding). Fluorescence of tyrosine has been used to monitorbinding of metals to β-amyloid [Garzon-Rodriguez W. et al., Bioorg. Med.Chem. Let., 9:2243-2248 (1999), which is hereby incorporated byreference]. A similar approach was taken to analyze binding of iron toα-synuclein. Tyrosine has a fluorescence emission spectrum that has apeak emission of 310 nm when excited at 280 nm, respectively (FIG. 2A).It was observed that binding of iron reduced the fluorescence of thetyrosines in α-synuclein. The result demonstrated that it is due tospecific binding to α-synuclein, because incubation of free tyrosinewith similar doses of FeCl₂ produces no quenching. Using the Prismprogram (GraphPad, San Diego, Calif.) to analyze the binding curves, weobserved that the data fit to a sigmoidal curve, with an IC₅₀=173 μM.The Hill coefficient was 1.0 (R²=1.0, P<0.0001), indicating one bindingsite for iron with no cooperativity (FIGS. 2A & B). α-Synuclein was alsotested for binding to other metals, including CuCl₂, ZnCl₂ and MgCl₂.Because the C-terminus of α-synuclein has a region with 7 glutamates and4 aspartates, which together could chelate iron, we created a deletionconstruct lacking the last 27 amino acids on α-synuclein and analyzedbinding of this construct. A C-terminal deletion construct ofα-synuclein AC₁₋₁₁₃ showed over a 4-fold reduction in iron binding, withan IC₅₀=726 μM (P<0.001) (FIG. 2B). This suggests that α-synuclein bindsto iron, and that the C-terminus contributes to iron-binding, but is notrequired for iron binding.

Example 8 Magnesium Protects Against Synuclein Aggregation

To monitor the binding profiles of Fe(II) and Mg(II) to α-synuclein invitro we used changes of tyrosine fluorescence. Tyrosine fluorescence isused as an indicator of changes in protein conformation or binding ofmetals. Excitation of tyrosine at 280 nm elicits fluorescence that peaksat 310 nm for monomeric tyrosine, and at 350-400 nm for tyrosinate ordimeric tyrosine. The fluorescence spectrum of α-synuclein yieldedfluorescence peaks at 310 and 400 nm. The peak at 400 nm cannot beexplained by the presence of dimeric tyrosine, due to dimerization ofα-synuclein, because dimerization of α-synuclein decreases thefluorescence at 400 nm, and tends to eliminate the peak at 400 nm.Moreover, gel electrophoresis and mass spectrometry of the α-synucleinshowed that the α-synuclein was monomeric. This suggests that the peakat 400 nm is due to tyrosinate, which could result from proton transferfrom the phenolic hydroxyl to aspartic or glutamic acid proteinacceptors. Incubating α-synuclein with increasing doses of Fe(II)rapidly reduced the fluorescence of tyrosines in α-synuclein at both 310and 400 nm. Plotting of the fluorescence quenching showed a sigmoidcurve, with an IC50=173 μM and a Hill coefficient of 1.0 (R2=1.0,P<0.0001), indicating one binding site or multiple binding sites withthe same affinity and no cooperativity. The actual affinity of iron forα-synuclein could be below 173 μM because tyrosine fluorescence is abetter indicator of conformation changes than absolute affinity.

The effect of magnesium on α-synuclein differed dramatically from thatof iron. Magnesium increased the fluorescence at 400 nm but did notaffect the fluorescence at 310 nm. Binding of magnesium to α-synucleinwas also striking because the tyrosine fluorescence showed a sharpstepwise increase between 60 and 80 uM of magnesium indicatingcooperative binding. The cooperative regulation of tyrosinefluorescence, specifically at 400 nm, suggests that magnesium causes aconformational change in synuclein differing from that induced by iron.Co-incubating magnesium with iron did not prevent iron-induced quenchingof α-synuclein tyrosine fluorescence suggesting that iron and magnesiumbind to different sites on α-synuclein.

We also examined how the A53T mutation in human α-synuclein affectedbinding of iron and magnesium. The A53T mutation did not change theapparent affinity of iron for α-synuclein, but did abolish theinteraction between magnesium and α-synuclein. Previous studies haveshown that A53T mutation changes the conformation of α-synuclein byincreasing its helical content (Polymeropoulos et al, Science,276:2045-7(1997)). These conformational changes might also reducebinding of magnesium to α-synuclein.

Knowing that magnesium and iron induce opposing changes in α-synucleintyrosine fluorescence, it is believed that binding of magnesium mightconvert α-synuclein to a conformation that resists aggregation perhapsby preventing β-pleated sheet formation. To test this, wildtyperecombinant α-synuclein was incubated with 0-3 mM FeCl₂ and 0 or 100 μMMgCl₂ for 24 hrs at 37° C., then immunoblotting was performed forα-synuclein and quantified aggregation by video densitometry. Weobserved a striking reduction in formation of high molecular weightaggregates of α-synuclein at all but the highest dose of iron (n=3,P<0.0001). Analyses using thioflavine-S to measure aggregation ofrecombinant α-synuclein also showed induction of α-synuclein aggregationby iron and inhibition of aggregation by magnesium under similarconditions. In separate experiments, we observed that magnesium alsoinhibited spontaneous aggregation of α-synuclein.

Since we did not observe interaction between magnesium and A53Tα-synuclein using tyrosine fluorescence, we tested whether aggregationof recombinant A53T α-synuclein was also insensitive to magnesium. Weincubated recombinant A53T α-synuclein with 0-3 mM FeCl₂ and 0 or 100 μMMgCl₂ for 24 hrs, then immunoblotted the α-synuclein and quantifiedaggregation by video densitometry. We observed that magnesium was unableto inhibit iron-induced aggregation of A53T α-synuclein. The inabilityof magnesium to inhibit aggregation of A53T α-synuclein is consistentwith the tyrosine fluorescence data suggesting that magnesium does notinteract with A53T α-synuclein.

The ability of magnesium to inhibit aggregation of wildtype α-synucleinin vitro suggests that it might also be able to inhibit aggregation inliving neurons. To test this, the inventors examined the ability ofmagnesium to inhibit α-synuclein aggregation in primary cortical neuronsgrown in cell culture. Primary cortical neurons were treated with 1 mMFeCl₂ plus 0-1.5 mM MgCl₂ for 3 days, the α-synuclein was immunublottedand the amount of aggregate determined by video densitometry. Treatmentwith increasing doses of magnesium from 0.2 to 1.5 mM produceddose-dependent reduction in formation of high molecular weightα-synuclein aggregates. Doses as low as 0.5 mM of magnesium producedstatistically significant reduction in formation of α-synuclein positiveaggregates. Magnesium exerted a similar aggregation-inhibiting effect onBE-M17 neuroblastoma cells over-expressing wild type α-synuclein. Incontrast, magnesium was unable to inhibit aggregate formation in BE-M17cells expressing the human A53T α-synuclein cDNA, which is consistentwith a hypothesis that magnesium inhibits α-synuclein aggregation bybinding to α-synuclein, inducing a conformational change.

The inventors also examined the amount of aggregates present using anantibody to ubiquitin. As set forth previously, the accumulation of highmolecular weight ubiquitin adducts correlates with the formation ofα-synuclein aggregates. Primary cortical neurons were treated asdescribed above and the aggregates were immunoprecipitated withanti-α-synuclein antibody and immunoblotted with anti-ubiquitinantibody. An increase in ubiquitination of α-synuclein upon treatmentwith 0.3 mM iron and 50 μM dopamine for 3 days was observed. However,treating the cells with magnesium produced a statistically significantdecrease in ubiquitin immunoreactivity with doses as low as 0.5 mM (n=3,P<0.0001). Thus, magnesium inhibits formation of ubiquitin positiveα-synuclein aggregates in primary neurons.

Next, whether magnesium also prevents formation of α-synuclein-positiveinclusions in BE-M17 neurons stably over-expressing wildtype α-synucleinwas investigated. The neurons were treated with 1 mM FeCl₂ plus 50 μMdopamine plus 0 or 1 mM MgCl₂ for 3 days, and then examined byimmunocytochemistry using anti-α-synuclein and anti-ubiquitinantibodies. Neurons treated with iron and dopamine showed abundantα-synuclein-positive inclusions, whereas neurons treated with iron,dopamine and magnesium showed little accumulation ofα-synuclein—positive inclusions.

Magnesium-induced inhibition of α-synuclein aggregation also correlatedwith neuroprotection, as measured by the MTT assay. BE-M17 neuroblastomacells stably transfected with vector or wildtype α-synuclein weretreated with FeCl₂ plus dopamine plus 0 or 0.8 mM MgCl₂ for 2 days, andthen viability was analyzed by the MTT assay (the doses were chosen tooptimize optical densities for the MTT assay). The cell lineover-expressing wildtype α-synuclein showed increased toxicity inresponse to the iron/dopamine treatment, which is consistent with theobservations above. Treatment with magnesium increased cell survival2.4-fold in the wildtype cell line but only 1.3-fold in the control cellline (compared to survival among cells treated with Fe²⁺/dopaminealone). This suggests that the cell line with more α-synuclein-relatedtoxicity had correspondingly more protection by magnesium.

How magnesium affected neurons from the central nervous system was alsoexamined. Primary cultures of cortical neurons were treated with 0.3 mMFeCl₂ plus 50 μM dopamine plus 0-2 mM MgCl₂ for 3 days (a relatively lowdose of iron was used to enable quantitation of neuronal processes). Toquantify the amount of neuroprotection provided by magnesium, thepercent of processes longer than 100 μm as a characteristic of cellviability was determined. Treating with iron plus dopamine lead toblunting of neuronal processes, reducing the number of neuronalprocesses over 100 μl by about 60% (n=5, P<0.001). Inclusions alsoformed in these neurons. However, neurons treated with iron, dopamine,and 1-2 mM magnesium showed only a 20% loss of processes, and did notexhibit any formation of α-synuclein-positive inclusions. These resultsare consistent with previous studies, indicating that magnesium iscytoprotective (Beal, M. F., Ann. Neurol. 31:119-30 (1992)).Furthermore, these results indicate that magnesium can preventα-synuclein aggregation in neurons of the central nervous system as wellas in neuronal cell lines.

In summary, magnesium inhibited both spontaneous and iron-inducedaggregation (FIG. 3). This could occur because binding of magnesium toα-synuclein converts α-synuclein to a conformation that resistsaggregation (FIG. 3B).

Interestingly, the dose at which magnesium exerts its anti-aggregatingeffects is similar to the therapeutic dose used to treat pre-eclampsia,for example. These doses of magnesium are therapeutically achievable.This could occur because binding of magnesium to α-synuclein convertsα-synuclein to a conformation that resists aggregation (FIG. 3B).

Example 9 Identification of Anti-α-Synuclein Peptides

Phage display (phage display kit was from New England Biolabs (Beverly,Mass.)) was used to specifically select for peptides that can inhibitiron binding. Phages were identified from libraries that bind to aminoacids 121-131 and amino acids 61-87 of α-synuclein and partially inhibitiron-induced aggregation of α-synuclein. The peptides were selectedusing the α-synuclein 121-131 and 61-87 peptides as the bait. One suchpeptide has the sequence SLKRLPK (SEQ ID NO: 7).

To demonstrate that the peptide SLKRLPK (SEQ ID NO: 7) bindsα-synuclein, the α-synuclein was absorbed to a plastic well, phage wasadded at dilutions of 1: 10, 1: 100, and 1: 1000, incubated 1 hour andwashed 5 times. Bound phage was detected by adding peroxidase coupledanti-phage antibody, incubating 1 hr, washing 5 times and detectingsignal using the peroxidase substrate ABTS. Bound phage produces a darkgreen signal that is measured as optical density with aspectrophotometer. Using these conditions, the phage containing theSLKRLPK (SEQ ID NO: 7) peptide gave OD's of 0.882, 0.844, and 0.480 atdilutions of 1: 10, 1: 100, and 1: 1000, respectively. By contrast,another phage that did not bind gave OD's of 0.019, −0.027, and −0.554respectively.

This peptide was also tested for its ability to inhibit iron-inducedα-synuclein aggregation in vitro. A 10-fold excess of the peptide wasable to inhibit α-synuclein aggregation by 38%, as determined bydensitometric quantification. This level of inhibition is extremelysignificant, given that if this were to translate into a 38% reductionin vivo it could slow the progression of PD by several years.

The peptide has the sequence “SLKRLPK” (SEQ ID NO: 7), and correspondsto a sequence expressed by a phage that shows particularly strongbinding to α-synuclein by ELISA. The sequence does not contain tyrosinesor tryptophans that can add to the fluorescence and complicate theanalyses. This phage was generated before we understood the importanceof iron for α-synuclein biochemistry (hence, the binding site was notspecifically targeted against iron binding). Even so, incubating a10-fold excess of peptide with α-synuclein reduces iron-inducedα-synuclein aggregation by 38%.

1. An isolated peptide that will bind to α-synuclein and inhibitaggregation of α-synuclein, said peptide consisting of a sequenceselected from: WRQTRKD (SEQ ID NO:1); HYAKNPI (SEQ ID NO:2); ATINKSL(SEQ ID NO:3); RRRGMAI (SEQ ID NO:4); TKHGPRK (SEQ ID NO:6); SLKRLPK(SEQ ID NO:7); RLRGRNQ (SEQ ID NO:8); WPFHHHR (SEQ ID NO:9); HLYHHKT(SEQ ID NO:10); THIHHPS (SEQ ID NO:11); or MMMMMRL (SEQ ID NO: 12). 2.The peptide of claim 1, wherein the peptide is SLKRLPK (SEQ ID NO:7),THIHHPS (SEQ ID NO:11), or MMMMMRL (SEQ ID NO:12).
 3. The peptide ofclaim 2, which is SLKRLPK (SEQ ID NO:7).
 4. A composition comprising apeptide of claim 1, and a pharmaceutically acceptable carrier.
 5. Thecomposition of claim 4, wherein the peptide is SLKRLPK (SEQ ID NO:7),THIHHPS (SEQ ID NO:11), or MMMMMRL (SEQ ID NO:12).
 6. The composition ofclaim 5, wherein the peptide is SLKRLPK (SEQ ID NO:7).
 7. A method fortreating a neurodegenerative disease that involves the formation of Lewybodies, comprising administering to a patient in need thereof one ormore of the peptides according to claim 1, whereby the presence of Lewybodies remains the same or is reduced.
 8. The method of claim 7, whereinthe one or more peptides is selected from: SLKRLPK (SEQ ID NO:7);THIHHPS (SEQ ID NO:11); or MMMMMRL (SEQ ID NO:12).
 9. The method ofclaim 7, wherein the peptide is SLKRLPK (SEQ ID NO:7).
 10. The method ofclaim 7, wherein the neurodegenerative disease is Parkinson's disease(PD), Alzheimer's disease (AD) diffuse Lewy body disease, mixed AD-PD,multiple system atrophy or Hallervorden-Spatz disease.
 11. The method ofclaim 7, wherein the neurodegenerative disease is Parkinson's disease.12. A method for inhibiting the formation of aggregates of α-synuclein,comprising treating the α-synuclein with a peptide according to claim 1.